Fibrin blood clot formation is mediated by a series of enzymatic reactions that occur on cellular and vascular surfaces. The coagulation proteins circulate in the blood as inactive proteins (zymogens) and upon stimulation are proteolyzed to generate active enzymes. Traditional coagulation models represent the series of reactions as a Y-shaped “cascade” with two separate pathways—the extrinsic and intrinsic pathway—that ultimately converge into a final common pathway (Macfarlane, Nature 202:498-9 (1964), Davie and Ratnoff, Science 145:1310-2 (1964)). Thrombin is the final enzyme formed in the coagulation cascade, and the rate of thrombin formation, as well as the amount of thrombin formed directly influences fibrin clot stability and structure (Wolberg, Blood Rev 21:131-42 (2007)).
Inappropriate thrombin generation can result in pathological blood clot formation, termed thrombosis. The treatment of patients with thrombosis almost always includes the administration of an anticoagulant therapeutic to impair procoagulant protein function and prevent blood coagulation. Researchers currently debate the optimal therapeutic target as the degree of anticoagulation required varies depending on the clinical indication. For example, lower levels of anticoagulation are desired for prophylactic treatment of high risk patients, while potent anticoagulation is required during surgical procedures, such as cardiopulmonary bypass (CPB), to treat thrombosis.
Genetic studies with knockout mice have been performed to study the role of each coagulation protein and pathway specific responses. Although Hemophilic mice (FVIII or FIX deficiency) have been extensively studied to discern the role of these proteins during in vivo coagulation, genetically null mice for TF, FVII, FX, and prothrombin are not viable, making similar studies unfeasible (Mackman, Arterioscier Thromb Vasc Biol 25: 2273-81 (2005)). Alternatively, inhibiting clotting proteins with currently available anticoagulant therapeutics can functionally remove the enzyme from the system and thereby clarify the role of these proteins in clot formation. Although small molecule anticoagulants have been generated toward a few coagulation enzymes (i.e., thrombin and FXa), it has been challenging to design similar compounds toward the upstream coagulation enzymes (i.e., FVIIa and FIXa). Thus, alternative classes of therapeutics that can be applied to inhibit all of the procoagulant proteins are needed to fully probe and directly compare the contributions of each pathway.
Aptamers, or single-stranded oligonucleotides, are nucleic acid ligands that bind specifically to their therapeutic targets with high affinity. Aptamers can be generated against target molecules, such as soluble coagulation proteins, by screening combinatorial oligonucleotide libraries for high affinity binding to the target (Ellington and Szostak, Nature 1990; 346: 8 18-22 (1990), Tuerk and Gold, Science 249:505-10 (1990). This selection method has been employed to generate modified RNA aptamers that bind specifically to coagulation factor VIIa (FVIIa) (Layzer and Sullenger, Oligonucleotides 17:1-11 (2007)), factor IXa (FIXa) (Rusconi et al, Nature 419: 90-4 (2002)), factor X (FXa) (Buddai et al, J Biol Chem 285:52 12-23 (2010)), or prothrombin (Layzer and Sullenger, Oligonucleotides 17:1-11 (2007), Bompiani et al, J Thromb Haemost 10:870-80 (2012). All of the aptamers bind to both the zymogen and enzyme form of the protein, and mechanistic studies with the FIXa, FXa, and prothrombin aptamers indicate that the aptamers bind a large surface area on the zymogen/enzyme that is critical for procoagulant protein-protein interactions (Buddai et al, J Biol Chem 285:52 12-23 (2010), Bompiani et al, J Thromb Haemost 10:870-80 (2012), Long et al, RNA 14:1-9 (2008), Sullenger et al, J Biol Chem 287:12779-86 (2012)). Moreover, two independent types of antidotes have been developed that can rapidly modulate aptamer anticoagulant function, which increases the safety profile of these anticoagulants (Rusconi et al, Nature 419: 90-4 (2002), Oney et al, Nat Med 15:1224-8 (2009)). Currently, an optimized version of the FIXa aptamer and its antidote are in phase two clinical trials for thrombotic indications (Cohen et al, Circulation 122:614-22 (2010), Povsic et al, Eur Heart J 32:2412-9 (2011)). The phase one clinical data indicate that this aptamer is well tolerated, safe, and can be rapidly modulated with the antidote (Dyke et al, Circulation 2006; 114:2490-7 (2006), Chan et al, Circulation 117:2865-74 (2008), Chan et al, J Thromb Haemost 6: 789-96 (2008)).
The present invention results, at least in part, from studies designed to compare the effects of the FVIIa, FIXa, FXa, and prothrombin anticoagulant aptamers in several in vitro clotting assays to assess the impact of inhibiting coagulation proteins in the different pathways. Additionally, the effects of simultaneously inhibiting two proteins within the same or different pathways by combining two aptamers were analyzed. These anticoagulant aptamers were also tested in a clinically relevant model of ex vivo extracorporeal circulation and their ability to be controlled with an antidote was assessed. The results demonstrate that aptamers are a unique class of anticoagulants that can be used individually or in combination, yet still safely controlled with antidotes.